D2-40, oncofetal M2A antigen

D2-40 was initially considered to be directed against the M2A antigen, a 40 kDa O-linked sialoglycoprotein found on the cell surface of testicular gonocytes, germ cell tumours lymphatic endothelium and mesothelial cells. Using a human podoplanin-Fc fusion protein, it has been shown that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses⁸.

D2-40 is immunoreactive with formalin-fixed paraffin embedded tissue.

Immunohistochemical expression

The lymphatic staining is in a thin linear pattern: unlike CD31 and CD34, blood vessel endothelium is negative.

The basal layer of sebaceous glands and outer root sheath of hair follicles stain.

Kaposi sarcoma stains, suggesting derivation from lymphatics.

D2-40

CD31

Lymphangioma

10/10⁶, 10/10⁷

5/10⁶, 5/10⁷

Haemangioma

0/10⁶ 0/22⁷

10/10⁶, 27/27⁷

Spindle cell haemangioma

1/5⁷

Glomus tumour

0/3⁶, 0/5⁷

0/3⁶, 0/5⁷

Angiolipoma

0/2⁶

2/2⁶

Pyogenic granuloma

0/2⁶

2/2⁶

Vascular malformation

0/2⁶

2/2⁶

Reactive angioendotheliomatosis

1/1

1/1⁷

Vascular transformation of lymph node

1/1⁷

1/1⁷

Dabska tumour

3/3⁷

3/3⁷

Haemangiopericytoma

0/1⁶

1/1 (Endothelial cells were positive and pericytes were negative.)⁶

Epithelioid haemangioendothelioma

0/1⁶, 1/10⁷

1/1⁶, 10/10⁷

Retiform haemangioendothelioma

0/1⁷

1/1⁷

Kaposiform haemangioendothelioma

0/1⁷

1/1⁷

Cutaneous Kaposi sarcoma

24/24 (cases included patch, plaque and nodular stages)⁶, 9/10⁷

24/24 (positivity was weak)⁶, 9/10⁷

Angiosarcoma

3/7⁶, 7/15⁷

7/7⁶, 15/15⁷

Neurofibroma

0/6⁶

Dermatofibroma

2/6⁶

Dermatofibrosarcoma protuberans

0/4⁶

Leiomyosarcoma

3/10⁶

Malignant fibrous histiocytoma

1/2⁶

Mesothelial immunoreactivity is membranous and, to a lesser extent, cytoplasmic. Other antibodies are shown for comparison.

D2-40

Calretinin

WT1

CK5/6

rate of positivity

mean percentage of cells staining in positive cases

Mesothelioma

epithelioid

33/33¹, 32/45 (the lower sensitivity may have been because this was a tissue microarray based study.)⁵, 84/112 (epithelioid mesotheliomas studied by TMAs)¹¹

92%¹

33/33¹, 104/112 (epithelioid mesotheliomas studied by TMAs)¹¹

19/33¹

29/32¹

biphasic

15/16¹, 96/162 (epithelioid areas of biphasic mesotheliomas studied by TMAs)¹¹, 39/120 (sarcomatoid areas of biphasic mesotheliomas studied by TMAs)¹¹

epithelioid cells: 90%, sarcomatous cells: 26%¹

16/16¹, 148/164 (epithelioid areas of biphasic mesotheliomas studied by TMAs)¹¹, 70/121 (sarcomatoid areas of biphasic mesotheliomas studied by TMAs)¹¹

10/16¹

13/16¹

sarcomatous

3/4¹, 11/46 (sarcomatoid mesotheliomas studied by TMAs)¹¹

52%¹

2/4¹, 25/46 (sarcomatoid mesotheliomas studied by TMAs)¹¹

0/4¹

1/4¹

Reactive pleura

27/28¹

71%¹

Pulmonary adenocarcinoma

2/31 (weak cytoplasmic staining)¹

13%¹

7/31¹

2/31¹

12/31¹

Ovarian serous carcinoma

17/26 (membranous staining)¹

33%¹

8/26¹

23/26¹

9/26¹

Renal cell carcinoma

Conventional

0/16¹

2/16¹

0/16¹

5/16¹

Papillary

5/13 (weak cytoplasmic staining)¹

43%¹

1/13¹

1/13¹

7/13¹

Chromophobe

0/5¹

1/5¹

1/5¹

1/5¹

Breast carcinoma

3/16 (weak cytoplasmic staining)¹

13%¹

Prostatic adenocarcinoma

0/11¹

Urothelial carcinoma

0/7¹

Calretinin has a greater sensitivity than does D2-40 in both epithelioid and sarcomatoid mesotheliomas but it is useful to combine them in a panel:

Sensitivities:

epithelioid

sarcomatoid

calretinin

0.91

0.57

D2-40

0.66

0.3

positivity for calretinin or D2-40

0.96

0.66

Another study evaluated podoplanin alongside D2-40²:

D2-40

Podoplanin

Mesothelioma

epithelioid

25/29 (better differentiated tumours show thick continuous membrane staining. In poorly differentiated tumours, membrane staining is discontinuous or staining is dot-like. Intracytoplasmic globoid staining may occur.)

25/29 (staining was seen in the same 25 cases that showed positivity with D2-40)

biphasic

4/5 (positivity confined to the epithelioid component)

4/5 (positivity confined to the epithelioid component)

sarcomatoid

0/6

0/6

Adenocarcinoma

lung

0/24

0/24

ovary

0/17

0/17

endometrium

0/5

0/5

breast

0/10

0/10

pancreas

0/5

0/5

stomach

0/5

0/5

colon

0/10

0/10

kidney

0/10

0/10

prostate

0/5

0/5

thyroid

0/3

0/3

Adenomatoid tumour

2/2 (staining is of the apical cell membrane)

2/2

Squamous carcinoma of lung

0/10

0/10

Angiosarcoma

2/5

2/5

Synovial sarcoma

biphasic

4/6 (positivity confined to the epithelioid component)

4/6 (positivity confined to the epithelioid component)

monophasic

0/6

0/6

This study was based on cell blocks prepared from serous effusions³. In cases where tumours stained, the pattern was membranous.

up to 10% of tumour cells staining

more than 10% of tumour cells staining

reactive mesothelium

153/153 (8 purely reactive, 145 mesothelial cells in the presence of malignancy)

malignant mesothelioma

2/32 (24 pleura, 8 peritoneal)

30/32 (24 pleura, 8 peritoneal)

malignant mesothelioma biopsies

12/12

ovarian carcinoma

85/169 (138 serous [including 23 primary peritoneal and 8 tubal], 3 clear cell, 3 endometrioid, 7 undifferentiated, 6 combined, 2 undefined: 129 peritoneal, 40 pleural)

13/169 (138 serous [including 23 primary peritoneal and 8 tubal], 3 clear cell, 3 endometrioid, 7 undifferentiated, 6 combined, 2 undefined: 129 peritoneal, 40 pleural)

ovarian microarray of biopsies

5/72 72 cores of primary and metastatic ovarian carcinoma from 23 patients)

9/72 72 cores of primary and metastatic ovarian carcinoma from 23 patients)

breast carcinoma

11/44 (40 pleura, 3 peritoneal)

2/44 (40 pleura, 3 peritoneal)

adenocarcinoma of unknown origin

3/14 (7 pleural, 7 peritoneal)

3/14 (7 pleural, 7 peritoneal)

lung carcinoma

2/6 (all pleural)

0/6 (all pleural)

Other cancers (4 oesophagus, 4 cervix uteri, 2 endometrial, 3 gastric, 1 colon, 1 pancreas, 1 prostate, 1 melanoma)

7/17 (2/2 cervical, 1/2 endometrial, 1/3 gastric, 2/4 oesophageal, 1/1 pancreatic, 1/1 prostatic and 1/1 melanoma were positive)

2/17 (2/2 cervical, 1/2 endometrial, 1/3 gastric, 2/4 oesophageal, 1/1 pancreatic, 1/1 prostatic and 1/1 melanoma were positive)

Primary cutaneous carcinomas are commonly positive while metastases to skin are negative:

primary skin tumours

Sebaceous carcinoma

10/10 (diffusely positive in 6 cases, focally positive in 4 cases)⁹, 2/2⁹

Squamous cell carcinoma

10/10 (diffusely positive in 8 cases, focally positive in 2 cases)⁹

Porocarcinoma

4/4 (diffusely positive in 1 case, focally positive in 3 cases)⁹, 2/2⁹

Trichilemmal carcinoma

4/4 (focally positive in all 4 cases)⁹

Sweat gland adenocarcinoma

1/1⁹

Adenoid cystic carcinoma

0/1⁹

Skin adnexal carcinoma NOS

4/4 (diffusely positive in 4 cases, focally positive in 4 cases)⁹, 0/1 (poorly differentiated adnexal carcinoma)⁹

Atypical adnexal tumour

1/1⁹

Benign primary adnexal tumour

38/46 (diffusely positive in 12 cases, focally positive in 26 cases. these cases consisted of 14 sebaceous adenomas, 15 syringomas, 9 hidradenomas, 7 eccrine poromas and 1 spiradenoma. sebaceous adenomas shows staining of the basaloid cells. )⁹, 6/6 (2 spiradenomas, 2 benign mixed tumours, 1 papillary hidradenoma, 1 nodular hidradenoma)⁹

Carcinoma metastatic to skin

0/15 (the metastases were from breast (6 cases), colon, lung, biliary tract, ovary, pancreas, thyroid, and one retroperitoneal spindle cell sarcoma.)⁹, 1/21 (0/6 lung, 0/5 breast, 0/2 colon, 1/1 renal, 0/1 gastric, 0/1 prostatic, 0/1 biliary, 0/1 ovarian, 0/1 bladder, 0/1 unknown primary)⁹

Adenomatoid tumour: 2/2¹²

Diagnostic utility

Diagnosis of lymphatic-derived tumours. Positivity may be helpful in differentiating Kaposi sarcoma from other vascular lesions.
Diagnosis of lymphatic invasion by tumours. Lymphatic invasion by breast carcinoma is predictive of distant recurrences and shortened survival¹⁰.
Differentiation of mesothelioma from adenocarcinoma⁴. The initial evidence as to its usefulness in the differentiation of mesothelioma from serous ovarian tumours is inconsistent. Like other mesothelial markers, it appears least useful in sarcomatous mesotheliomas.
Differentiation of primary skin adnexal carcinomas from adenocarcinomas metastatic to the skin⁹.

References

¹Chu AY, Litzky LA, Pasha TL, Acs G,Zhang PJ Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma. Mod Pathol 2005; 18:105-10

²Ordonez NG D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Hum Pathol 2005; 36:372-80

³Bassarova AV, Nesland JM,Davidson B. D2-40 is Not a Specific Marker for Cells of Mesothelial Origin in Serous Effusions. Am J Surg Pathol 2006; 30:878-82

⁴Ordonez NG. Immunohistochemical diagnosis of epithelioid mesothelioma: an update. Arch Pathol Lab Med 2005; 129:1407-14 FULL TEXT

⁵SienkoA, Zander DS, Killen D et al. D2-40 is a novel new marker of malignant mesothelioma (MM): tissue microarray study of 45 MM versus 409 lung carcinomas and primary non-mesothelial neoplasm of the pleura and chest wall. Mod Pathol 2005;18(suppl1):318A.

⁶Kahn HJ, Bailey D,Marks A. Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi's sarcoma and a subset of angiosarcomas. Mod Pathol 2002; 15:434-40

⁷Fukunaga M. Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours. Histopathology 2005; 46:396-402

⁸Schacht V, Dadras SS, Johnson LA, et al. Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors. Am J Pathol 2005; 166:913-21 FULL TEXT

⁹Liang H, Wu H, Giorgadze TA, et al. Podoplanin is a highly sensitive and specific marker to distinguish primary skin adnexal carcinomas from adenocarcinomas metastatic to skin. Am J Surg Pathol 2007; 31:304-10

¹⁰Arnaout-Alkarain A, Kahn HJ, Narod SA, et al. Significance of lymph vessel invasion identified by the endothelial lymphatic marker D2-40 in node negative breast cancer. Mod Pathol 2007; 20:183-91

¹¹Hinterberger M, Reineke T, Storz M, et al. D2-40 and calretinin - a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation. Mod Pathol 2007; 20:248-55

¹²Kuroda N, Toi M, Hiroi M, et al. Diagnostic pitfall of D2-40 in adenomatosid tumour. Histopathology 2007; 51:719-21

This page last revised 28.4.2007.

	D2-40	CD31
Lymphangioma	10/10⁶, 10/10⁷	5/10⁶, 5/10⁷
Haemangioma	0/10⁶ 0/22⁷	10/10⁶, 27/27⁷
Spindle cell haemangioma	1/5⁷
Glomus tumour	0/3⁶, 0/5⁷	0/3⁶, 0/5⁷
Angiolipoma	0/2⁶	2/2⁶
Pyogenic granuloma	0/2⁶	2/2⁶
Vascular malformation	0/2⁶	2/2⁶
Reactive angioendotheliomatosis	1/1	1/1⁷
Vascular transformation of lymph node	1/1⁷	1/1⁷
Dabska tumour	3/3⁷	3/3⁷
Haemangiopericytoma	0/1⁶	1/1 (Endothelial cells were positive and pericytes were negative.)⁶
Epithelioid haemangioendothelioma	0/1⁶, 1/10⁷	1/1⁶, 10/10⁷
Retiform haemangioendothelioma	0/1⁷	1/1⁷
Kaposiform haemangioendothelioma	0/1⁷	1/1⁷
Cutaneous Kaposi sarcoma	24/24 (cases included patch, plaque and nodular stages)⁶, 9/10⁷	24/24 (positivity was weak)⁶, 9/10⁷
Angiosarcoma	3/7⁶, 7/15⁷	7/7⁶, 15/15⁷
Neurofibroma	0/6⁶
Dermatofibroma	2/6⁶
Dermatofibrosarcoma protuberans	0/4⁶
Leiomyosarcoma	3/10⁶
Malignant fibrous histiocytoma	1/2⁶

		D2-40		Calretinin	WT1	CK5/6
		rate of positivity	mean percentage of cells staining in positive cases	Calretinin	WT1	CK5/6
Mesothelioma	epithelioid	33/33¹, 32/45 (the lower sensitivity may have been because this was a tissue microarray based study.)⁵, 84/112 (epithelioid mesotheliomas studied by TMAs)¹¹	92%¹	33/33¹, 104/112 (epithelioid mesotheliomas studied by TMAs)¹¹	19/33¹	29/32¹
	biphasic	15/16¹, 96/162 (epithelioid areas of biphasic mesotheliomas studied by TMAs)¹¹, 39/120 (sarcomatoid areas of biphasic mesotheliomas studied by TMAs)¹¹	epithelioid cells: 90%, sarcomatous cells: 26%¹	16/16¹, 148/164 (epithelioid areas of biphasic mesotheliomas studied by TMAs)¹¹, 70/121 (sarcomatoid areas of biphasic mesotheliomas studied by TMAs)¹¹	10/16¹	13/16¹
	sarcomatous	3/4¹, 11/46 (sarcomatoid mesotheliomas studied by TMAs)¹¹	52%¹	2/4¹, 25/46 (sarcomatoid mesotheliomas studied by TMAs)¹¹	0/4¹	1/4¹
Reactive pleura		27/28¹	71%¹
Pulmonary adenocarcinoma		2/31 (weak cytoplasmic staining)¹	13%¹	7/31¹	2/31¹	12/31¹
Ovarian serous carcinoma		17/26 (membranous staining)¹	33%¹	8/26¹	23/26¹	9/26¹
Renal cell carcinoma	Conventional	0/16¹		2/16¹	0/16¹	5/16¹
	Papillary	5/13 (weak cytoplasmic staining)¹	43%¹	1/13¹	1/13¹	7/13¹
	Chromophobe	0/5¹		1/5¹	1/5¹	1/5¹
Breast carcinoma		3/16 (weak cytoplasmic staining)¹	13%¹
Prostatic adenocarcinoma		0/11¹
Urothelial carcinoma		0/7¹

Sensitivities:	epithelioid	sarcomatoid
calretinin	0.91	0.57
D2-40	0.66	0.3
positivity for calretinin or D2-40	0.96	0.66

		D2-40	Podoplanin
Mesothelioma	epithelioid	25/29 (better differentiated tumours show thick continuous membrane staining. In poorly differentiated tumours, membrane staining is discontinuous or staining is dot-like. Intracytoplasmic globoid staining may occur.)	25/29 (staining was seen in the same 25 cases that showed positivity with D2-40)
	biphasic	4/5 (positivity confined to the epithelioid component)	4/5 (positivity confined to the epithelioid component)
	sarcomatoid	0/6	0/6
Adenocarcinoma	lung	0/24	0/24
	ovary	0/17	0/17
	endometrium	0/5	0/5
	breast	0/10	0/10
	pancreas	0/5	0/5
	stomach	0/5	0/5
	colon	0/10	0/10
	kidney	0/10	0/10
	prostate	0/5	0/5
	thyroid	0/3	0/3
Adenomatoid tumour		2/2 (staining is of the apical cell membrane)	2/2
Squamous carcinoma of lung		0/10	0/10
Angiosarcoma		2/5	2/5
Synovial sarcoma	biphasic	4/6 (positivity confined to the epithelioid component)	4/6 (positivity confined to the epithelioid component)
Synovial sarcoma	monophasic	0/6	0/6

	up to 10% of tumour cells staining	more than 10% of tumour cells staining
reactive mesothelium		153/153 (8 purely reactive, 145 mesothelial cells in the presence of malignancy)
malignant mesothelioma	2/32 (24 pleura, 8 peritoneal)	30/32 (24 pleura, 8 peritoneal)
malignant mesothelioma biopsies		12/12
ovarian carcinoma	85/169 (138 serous [including 23 primary peritoneal and 8 tubal], 3 clear cell, 3 endometrioid, 7 undifferentiated, 6 combined, 2 undefined: 129 peritoneal, 40 pleural)	13/169 (138 serous [including 23 primary peritoneal and 8 tubal], 3 clear cell, 3 endometrioid, 7 undifferentiated, 6 combined, 2 undefined: 129 peritoneal, 40 pleural)
ovarian microarray of biopsies	5/72 72 cores of primary and metastatic ovarian carcinoma from 23 patients)	9/72 72 cores of primary and metastatic ovarian carcinoma from 23 patients)
breast carcinoma	11/44 (40 pleura, 3 peritoneal)	2/44 (40 pleura, 3 peritoneal)
adenocarcinoma of unknown origin	3/14 (7 pleural, 7 peritoneal)	3/14 (7 pleural, 7 peritoneal)
lung carcinoma	2/6 (all pleural)	0/6 (all pleural)
Other cancers (4 oesophagus, 4 cervix uteri, 2 endometrial, 3 gastric, 1 colon, 1 pancreas, 1 prostate, 1 melanoma)	7/17 (2/2 cervical, 1/2 endometrial, 1/3 gastric, 2/4 oesophageal, 1/1 pancreatic, 1/1 prostatic and 1/1 melanoma were positive)	2/17 (2/2 cervical, 1/2 endometrial, 1/3 gastric, 2/4 oesophageal, 1/1 pancreatic, 1/1 prostatic and 1/1 melanoma were positive)